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N Kaplowitz

Publications and source records attributed to N Kaplowitz.

At least 55 records · Page 3Linked to original sources
PubMed1996

Identification of a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium.

PURPOSE: To determine whether glutathione (GSH) transporter(s) other than the previously identified rat canalicular GSH transporter (RcGshT) is present in the lens. METHODS: Poly (A) +RNA isolated from rat and guinea pig lens cortex and epithelium was injected into Xenopus laevis oocytes. The effect of sodium removal was determined by measuring cell-associated radioactivity in lenticular epithelium or cortex mRNA injected oocytes (pretreated with acivicin to inhibit gamma glutamyltranspeptidase) after 1 hour of incubation in NaCl medium or choline chloride (Na(+)-free) medium containing tracer GSH (plus unlabeled GSH). The effect of 2 mM bromosulfophthalein-GSH (BSP-GSH) on GSH uptake in the lens epithelium and cortex in NaCl medium at two GSH concentrations also was determined. The molecular form of uptake of GSH in lens epithelial mRNA-injected oocytes was examined by high-performance liquid chromatography. Western blot analysis was performed to study the presence of RcGshT in the cortex and epithelium. RESULTS: Oocytes injected with mRNA from rat and guinea pig lens epithelium and cortex compartments expressed GSH transport. High-performance liquid chromatography confirmed that epithelial uptake was as intact GSH under conditions of inhibition of GSH synthesis with dl-buthionine sulfoximine. The mean GSH uptake (nmol/oocyte per hour) in epithelial mRNA-injected oocytes was significantly reduced (P < 0.01, n = 4 oocyte preparations) under Na(+)-free conditions compared to NaCl medium at 0.05 mM and 2 mM GSH in the medium. Uptake in cortical mRNA-injected oocytes was unaffected by Na+ removal. Lens epithelial uptake exhibited a strong inhibition by BSP-GSH at 0.05 mM (55%) and 2 mM (64%), whereas cortical uptake was unaffected by BSP-GSH. Western blot analysis identified RcGshT in the cortical and epithelial regions. CONCLUSIONS: Results from the current study provide strong evidence for the presence of a hitherto unreported Na(+)-dependent, BSP-GSH inhibitable GSH transporter in the lens epithelium, which may mediate concentrative, basolateral uptake of aqueous GSH consistent with in situ eye perfusion studies. The Na(+)-independent, BSP-GSH insensitive RcGshT may function as an apical GSH effluxer in lens epithelium and in mediating concentration gradient driven inward GSH movement by uptake-efflux in the lens cortex.

Animals↗
PubMed1995

Evidence that the rat hepatic mitochondrial carrier is distinct from the sinusoidal and canalicular transporters for reduced glutathione. Expression studies in Xenopus laevis oocytes.

Mitochondrial GSH derives from a mitochondrial transport system (RmGshT), which translocates cytosol GSH into the mitochondrial matrix. Mitochondria of oocytes, isolated 3-4 days after microinjection of total liver mRNA, expressed a RmGshT compared with water-injected oocytes. The expressed RmGshT exhibited similar functional features as reported in isolated mitochondria of rat liver such as ATP stimulation, inhibition by glutamate, and insensitivity to inhibition by sulfobromophthalein-glutathione (BSP-GSH) and S-(2,4-dinitrophenyl)glutathione (DNP-GSH). The expressed RmGshT is localized in the inner mitochondrial membrane since expression is still observed in mitoplasts prepared from total liver mRNA-injected oocytes. Fractionation of poly(A)+ RNA identified a single mRNA species of approximately 3-3.5 kilobases encoding for the RmGshT, which was stimulated by ATP and inhibited by glutamate but not by BSP-GSH or DNP-GSH. Microinjection of this fraction did not lead to expression of plasma membrane GSH transport in intact oocytes, and conversely, oocytes microinjected with cRNA for rat liver sinusoidal GSH transporter (RsGshT) or rat liver canalicular GSH transporter (RcGshT) did not express mitochondrial GSH transport activity. Thus, our results show the successful expression of the rat hepatic mitochondrial GSH carrier, which is different from RsGshT and RcGshT, and provide the strategic basis for the cloning of this important carrier.

Animals↗
PubMed1995

Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.

Using the Xenopus oocyte expression system, we previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an approximately 2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA was cloned and characterized as the putative rat canalicular GSH transporter. Starting with a cDNA library constructed from the approximately 2.5-kb fraction, we have now isolated a single clone that leads to expression of a BSP-GSH- and cystathionine-inhibitable GSH transporter activity with Km approximately 3 mM characteristic of the sinusoidal GSH transporter. The cDNA for the rat sinusoidal GSH transporter-associated polypeptide (RsGshT) is 2733 bases with an open reading frame of 1059 nucleotides encoding a polypeptide of 353 amino acids (39,968 Da) with two putative membrane-spanning domains. No identifiable homologies were found in searching various data bases. An approximately 40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single approximately 2.8-kb transcript in rat and human liver with negligible hybridization signal in other organs. The abundance of mRNA for RsGshT did not increase with phenobarbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristic of sinusoidal GSH secretion and thus indicate that RsGshT either encodes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.

Amino Acid Sequence↗
PubMed1995

Feeding S-adenosyl-L-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes.

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-L-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and periportal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 +/- 0.7 to 7.3 +/- 0.6 and 2.1 +/- 0.3 to 6.1 +/- 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.

Administration, Oral↗
PubMed1995

Microcystin uptake and inhibition of protein phosphatases: effects of chemoprotectants and self-inhibition in relation to known hepatic transporters.

The microcystins (Mcyst) are cyclic peptide hepatotoxins produced by cyanobacteria. They are chemically very stable and represent a public health threat when they occur in water bodies used for human consumption. Mice injected ip with Mcyst (LD50 50-100 micrograms/kg) accumulate Mcyst in the liver and die within 2-4 hr with massive intrahepatic hemorrhage. Pretreatment of mice with cyclosporin A (CP), rifamycin (Rif), trypan blue (TB), and trypan red (TR) protected the animals from a lethal dose of the toxin. The studies reported here using the freshly isolated rat hepatocyte model were undertaken in order (1) to evaluate the contribution of Mcyst transport in hepatocytes to the mechanism of chemoprotection for Mcyst in vivo toxicity by CP, Rif, TB, and TR and (2) to better characterize the hepatic Mcyst transporter in this model and determine its relationship to other bile acid/organic anion transporters that have already been fully described. Incubations with 125I-Mcyst were used to measure Mcyst uptake and accumulation in hepatocytes. It has been shown that at the cellular level Mcyst binds to and inhibits protein phosphatases 1 and 2A (PP) at nanomolar concentrations. Lethal doses of Mcyst in mice resulted in rapid profound inhibition of hepatic PP activity. PP activity was also inhibited in hepatocytes incubated with 100-500 nM Mcyst. PP inhibition in these studies was used as a marker of metabolic effects of the toxin. The chemoprotectants CP (5 microM), Rif (50 microM), TR (20 microM), and TB (20 microM) decreased accumulation of Mcyst (320 nM, a toxic concentration) after 30 min incubation to 37, 26, 30, and 66%, respectively, of that of cells treated with Mcyst only. Inhibition of PP activity in these cells was decreased. Inhibition of PP activity in hepatocytes was also decreased by known inhibitors of Mcyst transport: 50 microM of the bile acids cholate and taurocholate (TC) and 50 microM sulfobromophthalein. For all compounds tested the amount of Mcyst accumulated in the hepatocytes correlated qualitatively with the extent of PP inhibition. From these results it can be concluded that inhibition of Mcyst uptake by hepatocytes is the most likely mechanism of chemoprotection for Mcyst in vivo toxicity for TR, TB, CP, and Rif. Uptake of Mcyst was unaffected by changes in the ionic composition of the uptake buffer but was significantly decreased when PP activity of hepatocytes was inhibited by preincubation with Mcyst.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes.

Mitochondria are an important source of reactive oxygen intermediates because they are the major consumers of molecular oxygen in cells. Respiration is associated with toxicity, which is related to the activation of oxygen to reactive intermediates. The purpose of the present study was to examine the role of reduced glutathione (GSH) in the maintenance of mitochondrial functions during oxidative stress induced through selective inhibition of the complex III segment of the electron transport chain. Hydrogen peroxide monitored by the fluorescence of dichlorofluorescein increased in a time- and dose-dependent manner on incubation of mitochondria with antimycin A (AA), an inhibitor of complex III. However, blockade of complex I or II with rotenone or thenoyltrifluoroacetone, respectively, did not result in accumulation of hydrogen peroxide. Depletion of mitochondrial GSH to 10-20% of control by preincubation with diethylmaleate (0.8 mM) or ethacrynic acid (250 microM) also increased dichlorofluorescein and malondialdehyde levels and resulted in an additional (2-3-fold) increase after AA. Similar results were obtained when mitochondrial GSH depletion was produced by treatment with buthionine L-sulfoximine before mirochondria isolation. The endogenous oxidative stress induced by AA was accompanied by a moderate loss of activity of ATPase complex (77% of control) and complex IV of respiration (75% of control), which was accentuated after depletion of mitochondrial GSH (51% and 45% of control, respectively). Similar results were observed in isolated hepatocytes in which depletion of mitochondrial GSH and AA led to peroxidation and mitochondrial dysfunction. In addition, with electrophoretic mobility shift assay of the transcription factor nuclear factor-kappa B (NF-kappa B), we detected its activation in response to AA (2-3-fold). Depletion of mitochondrial GSH in hepatocytes (20% of control) led to further enhancement of NF-kappa B activation (2-4-fold), which correlated with generation of hydrogen peroxide. Thus, our results suggest that GSH protects mitochondria against the endogenous oxidative stress produced at the ubiquinone site of the electron transport chain. Mitochondrial GSH depletion potentiates oxidant-induced loss of mitochondrial functions. Oxidant stress in mitochondria can promote extramitochondrial activation of NF-kappa B and therefore may affect nuclear gene expression.

Animals↗
PubMed1995

Molecular characterization of a reduced glutathione transporter in the lens.

PURPOSE: To characterize glutathione (GSH) transporter in the lens. METHODS: Poly (A) +RNA isolated from bovine lens was injected into Xenopus laevis oocytes. Oocytes were incubated for 1 hour in either NaCl or sucrose medium containing tracer GSH, and cell-associated radioactivity was determined. Glutathione efflux was determined in lens mRNA injected oocytes preloaded with GSH. Relationship of lens GSH transporter to the two recently cloned sodium-independent hepatic membrane GSH transporters was studied by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Bovine lens mRNA also was probed for gamma glutamyl transpeptidase (GGT) by RT-PCR. RESULTS: Uptake of tracer 35S-GSH could be demonstrated in X. laevis oocytes injected with poly (A) +RNA from bovine lens. Glutathione transport was carrier mediated (Km approximately 1.3 mM) and was sodium independent. High-performance liquid chromatography confirmed that the molecular form of uptake was predominantly (> 98%) as it was for GSH. Poly (A) +RNA-injected oocytes preloaded with 16.5 nmol GSH-oocyte showed GSH efflux at a rate of 2.6 nmol/oocyte per hour. When bovine lens poly (A) +RNA was hybridized with the cDNA probe for the sodium-independent rat canalicular GSH transporter (RcGshT), the transcript for RcGshT was observed. RT-PCR confirmed the presence of RcGshT and showed the absence of rat sinusoidal GSH transporter (RsGshT) and GGT mRNA in rat lens. CONCLUSIONS: The authors have demonstrated for the first time that lens contains mRNA for RcGshT and expresses a low-affinity GSH transporter in oocytes. Glutathione efflux from the apical side of the anterior epithelium and progressive uptake, and inward efflux into cortical fibers, might be explained by expression of RcGshT alone or in combination with as yet unidentified GSH transporters.

Animals↗
PubMed1995

Mechanisms of drug-induced liver disease.

The liver is the main metabolizing organ in the body for drugs and toxins. The liver is therefore exposed to relatively high levels of electrophilic metabolites and free radicals that may induce toxicity. Furthermore, the liver performs many vital functions that may be disrupted by toxic metabolites. Despite the high exposure to reactive metabolites, drug-induced toxicity is relatively uncommon because of the redundancy of the detoxification systems present in the liver.

Animals↗
PubMed1994

Evidence for transcapillary transport of reduced glutathione in vascular perfused guinea-pig brain.

Using a vascular brain perfusion model in the guinea-pig, the net uptake of [35S]-GSH by the brain was found to be linear and similar in various regions during 10 min perfusion. Dual labeled [35S and 3H] GSH taken up by the brain had the same isotope ratio as the injected stock whether or not gamma- glutamyl transferase was inhibited. Greater than 96% of brain uptake of [35S]-cysteine-labeled GSH and [3H]-glycine-labeled GSH were in intact form. Transcytosis of [35S]-GSH from lumen into brain parenchyma was demonstrated using a capillary depletion technique. Both GSH and GSH-monoethyl ester inhibited [35S]-GSH transport. Thus, we have demonstrated blood-brain barrier extraction of circulating GSH in a brain perfusion model, and the transcytosis of intact GSH into the brain parenchyma without breakdown.

Animals↗
PubMed1994

Role of BSP/bilirubin binding protein and bilitranslocase in glutathione uptake in rat basolateral liver plasma membrane vesicles.

Glutathione (GSH) efflux in the liver is mediated by carrier proteins sensitive to membrane potential (electrogenic), and it is inhibited by organic anions. The hepatic uptake of tetrabromosulfophthalein (BSP) is also a carrier-mediated function accomplished at least by two different transport mechanisms, bilitranslocase (BTL) and BSP/Bilirubin Binding Protein (BBBP). The two proteins operate in parallel, the former operating electro-genically, the latter being insensitive to membrane potential (electroneutral). To investigate the relationship between transport mechanisms for GSH and organic anions, the initial uptake rate of 35S-labelled BSP into basolateral rat liver plasma membranes was measured in the presence of external or intravesicular GSH. Electrogenic and electroneutral BSP uptake were neither cis-inhibited nor trans-stimulated by GSH. 3H glycine-GSH uptake in LPMV was not modified by pre-incubating LPMV with anti-BTL or anti-BBBP antibodies. These data indicate that GSH hepatic uptake is mediated neither by BBBP nor BTL.

Animals↗
PubMed1994

Blood-to-lens transport of reduced glutathione in an in situ perfused guinea-pig eye.

Transport mechanisms of reduced glutathione (GSH) in intact eye are poorly understood. In this study, an in situ vascular eye perfusion (VEP) model was used to characterize the transport kinetics of circulating GSH into the aqueous humor and lens in guinea pigs. Radiolabeled [35S]GSH or [3H]GSH and [14C]sucrose (an extracellular space marker) were exposed to the blood-aqueous barrier up to 10 min, and uptake of tracers by the aqueous humor and lens was determined in the presence of different concentrations of unlabeled peptide as GSH, a gamma-glutamyl compound as a derivative of GSH (GSH monoethyl ester), and an inhibitor of gamma-glutamyl transpeptidase (GGT) activity. Plasma-aqueous and aqueous-lens compartmental unidirectional transport constant, K(in), and the initial rapid volume of distribution, Vi, were estimated by multiple-time-point graphic analysis. Our results indicated that both labeled GSH and sucrose entered the aqueous humor slowly at comparable rates with respective blood-aqueous K(in) values of 1.34 +/- 0.12 and 1.25 +/- 0.08 min-1 x 10(3). In contrast to blood-aqueous transport, GSH uptake by the lens was rapid, and the respective aqueous-lens K(in) values for labeled GSH and sucrose were 79.3 +/- 4.1 and 3.5 +/- 0.7 min-1 x 10(3). Over 94% of plasma-derived GSH remained in its original molecular form of GSH in the lens, during the 10 min perfusion both with and without the GGT inhibitor, serine borate. The amount of [35S]GSH in lens anterior epithelium (dpm mg-1) was more than three times that of aqueous humor (dpm microliter-1) within only 10 min of VEP. There was also significant accumulation of [35S]GSH in the interior cortex, as indicated by 10-min cortex/aqueous ratio of 0.65. A specific GGT independent GSH transport system was demonstrated in the lens in situ, with a Km of 26 +/- 3 microM, and Vmax of 34 +/- 3 pmol min-1 g-1 of whole lens tissue water. The lenticular influx of GSH was inhibited by GSH monoethyl ester with an affinity that was half that for GSH. It is concluded that: (a) uptake of plasma-derived GSH into the aqueous humor is by simple diffusion, and (b) cellular uptake of GSH by the lens is carrier-mediated via mechanism that is separate from the transpeptidation metabolic pathway.

Animals↗
PubMed1994

Distribution of 3 alpha-hydroxysteroid dehydrogenase (bile acid binder) in rat small intestine: comparison with glutathione S-transferase subunits.

We identified and quantitated the Y' bile acid binder, i.e., 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), in rat small intestinal mucosa, and compared its longitudinal distribution with that of glutathione S-transferases (GST). The enzyme activity of 3 alpha-HSD in intestinal mucosa was approximately one-third of that in liver, and it had similar activity in the proximal, middle, and distal portions of the intestine. Immunoreactive protein corresponding to hepatic bile acid binder was detected in all segments of rat small intestine mucosal cytosol by Western blot analysis. There was no significant difference in the concentration of bile acid binder, assayed by enzyme-linked immunosorbent assay (ELISA), between the proximal and the distal intestine, this being 2.93 +/- 0.03 and 3.29 +/- 0.95 nmol/g tissue, respectively (mean +/- SD of four animals). On the other hand, the concentration of GST 1-1 showed sharp longitudinal decline and that of GST 3-4 was negligible in the small intestine, as we previously reported, indicating that bile acid binder was a prominent cytosol binding protein in the distal intestine. These results suggested the possible role of bile acid binder in the intracellular transport of bile acids in the ileum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics.

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

Amino Acid Sequence↗
PubMed1994

Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.

Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease.

Animals↗
PubMed1994

Specificity and directionality of thiol effects on sinusoidal glutathione transport in rat liver.

In rats the sinusoidal glutathione (GSH) carrier transports GSH bidirectionally, and its activity is influenced by the thiol-disulfide status; the Vmax of sinusoidal GSH efflux was increased by dithiothreitol (DTT) and decreased by cystine. In the present work we examined the specificity and directionality of the thiol effect. Using in situ perfused livers, we found that 1 mM DTT and other dithiols, including 1,2-ethanedithiol, 1,3-propanedithiol, and 1,4-butanedithiol, stimulated sinusoidal GSH efflux by 200-500% but dihydrothioctic acid, which is negatively charged, had no effect. Uncharged or positively charged monothiols (2 mM), such as dimercaprol, monothioglycerol, 2-mercaptoethanol, 3-mercapto-2-butanol, 1-mercapto-2-propanol, and cysteamine, also exerted a stimulatory effect on sinusoidal GSH efflux. In contrast, monothiols containing a negatively charged substituent, such as penicillamine, captopril, N-acetylcysteine, mercaptopropionylglycine, mercaptoethanesulfonic acid, mercaptoacetic acid, and mercaptopropionic acid, had no effect. The thiol moiety was essential for activity, inasmuch as ethanol, propanol, propanediol, and glycerol had no effect on sinusoidal GSH efflux. The effect of DTT or cystine pretreatment (2 mM or 0.5 mM, respectively, for 30 min) on GSH uptake was then examined using cultured rat hepatocytes. The linear rate of [35S]GSH uptake and the concentration dependence were measured after cells were pretreated with acivicin (0.5 mM, for 15 min) and buthionine sulfoximine (10 mM, 15 min), to prevent breakdown and resynthesis of GSH from precursors, respectively. Uptake buffer also contained 20 mM alpha-(methylamino)isobutyric acid and 20 mM threonine (inhibitors of amino acid transport systems A and ASC, respectively), to prevent uptake of cysteine. Pretreatment with DTT decreased the Vmax of GSH uptake by approximately 50% (control Vmax value, 24 nmol/10(6) cells/30 min), whereas the Km remained unaffected (approximately 8 mM). Cystine pretreatment had no influence on GSH uptake but inhibited efflux. In conclusion, the presence of at least one thiol group and the absence of negative charge are required to stimulate sinusoidal GSH efflux. The direction of GSH transport is modulated by the thiol-disulfide status, so that thiol reduction changes the GSH transporter from a bidirectional GSH transporter into a preferentially unidirectional (outward) transporter by inhibiting uptake while stimulating efflux and thiol oxidation favors inward transport by inhibiting only efflux.

Analysis of Variance↗
PubMed1993

Selective induction by phenobarbital of the electrogenic transport of glutathione and organic anions in rat liver canalicular membrane vesicles.

Glutathione is excreted into bile via a low affinity, electrogenic, ATP-independent transport system which is cis-inhibited and trans-stimulated by certain organic anions (Fernández-Checa, J. C., Takikawa, H., Horie, T., Ookhtens, M., and Kaplowitz N. (1992) J. Biol. Chem. 267, 1667-1673). This transport system differs from the sinusoidal carrier in several respects, such as affinity for transport and inhibitor specificity. Another differential aspect is the selective increase by phenobarbital pretreatment of GSH excretion into bile without changing the sinusoidal release into blood. To determine if phenobarbital induces the GSH transporter in the canalicular membrane and if this is reflected in the induction of organic anion transport, we have used rat liver canalicular (cLPM) and sinusoidal (bLPM) enriched membrane vesicles from liver of control (saline) and phenobarbital-treated rats. cLPM vesicles prepared from phenobarbital-pretreated rats exhibited a significant, 46% increase in Vmax for transport (9.02 +/- 0.3 versus 6.17 +/- 0.5 nmol/mg/15 s) without a change in the Km for GSH transport (14.0 +/- 1.1 versus 16.7 +/- 2.7 mM, respectively). Kinetic parameters for GSH transport in bLPM vesicles remained unchanged after phenobarbital treatment versus control (Vmax, 4.67 +/- 0.2 versus 4.77 +/- 0.2 nmol/mg/15 s; Km, 7.79 +/- 0.8 versus 6.95 +/- 0.8 mM, respectively). Phenobarbital treatment increased the electrogenic transport of [35S]sulfobromophthalein (BSP) (5 and 50 microM) but not the electrogenic uptake of [14C] glycocholic acid (10 and 200 microM). In addition, the ATP-dependent transport of [35S]BSP, [3H]leukotriene C4, and [14C]glycocholic acid into cLPM vesicles was not altered by phenobarbital treatment. The ATP-independent transport of [35S]BSP in cLPM was cis-inhibited and trans-stimulated by GSH, supporting the view that BSP and GSH share a common multispecific transporter. Thus, among the various canalicular transport systems, the multispecific electrogenic organic anion and GSH transport system is selectively induced by phenobarbital treatment.

Adenosine Triphosphate↗
PubMed1993

Expression of rat liver reduced glutathione transport in Xenopus laevis oocytes.

We have studied the expression of the hepatic GSH transport system in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA resulted in the functional expression of the GSH transport system determined as GSH efflux from GSH loaded oocytes. Expression required 3-5 days to process the liver mRNA. Methionine, cystathionine, and sulfobromophthalein (BSP)-GSH inhibited the efflux of GSH from liver mRNA-injected oocytes according to their known cis or transactions on hepatocytes, namely BSP-GSH from inside and methionine and cystathionine from outside. The expressed hepatic GSH transport system also mediated the uptake of intact GSH into the oocyte, consistent with the bidirectional operation of this facilitative transporter. The uptake of GSH into mRNA-injected oocytes was inhibited by BSP-GSH in chloride-free conditions. Finally, two different mRNA size fractions encoded for hepatic GSH transport activity (uptake or efflux): a 2.0-2.5-kilobase size class, which expressed GSH transport (uptake or efflux) completely inhibited by BSP-GSH (compatible with sinusoidal GSH transport), and a 3.5-4.0-kilobase size class, which expressed GSH transport (uptake or efflux) not inhibited by BSP-GSH. These results demonstrate that hepatic GSH transport can be expressed in Xenopus oocytes and mRNA of two distinct sizes encode for GSH transporters.

Animals↗